Not applicable.
Significant research has been conducted in recent years on the use of biological agents to increase agricultural productivity and efficiency. Biological control based on the use of microorganisms to suppress plant pests offers an attractive alternative to chemical pesticides which are less favored than they have previously been because of concerns about human health and environmental quality. Several screening programs have been used before to isolate biological agents which are effective in the laboratory or in the field to combat pests or facilitate plant growth.
xe2x80x9cBiological controlxe2x80x9d or xe2x80x9cbiocontrolxe2x80x9d is defined as pathogen suppression by the use of a second organism. Mechanisms of biological control are diverse. For example, certain bacteria have been examined for their usefulness in biological control of root rot in alfalfa. It is believed that control is obtained by competition or antagonism between the bacteria and the fungi on the surface of the alfalfa roots. In contrast, a toxin produced by one species of bacteria may be used to control another species of bacteria or other microorganism that appears as a pathogen. Bacterially produced antibiotics are examples of such toxins. The toxin can be isolated from the species producing it and administered directly, as is the common procedure with penicillin, or the species itself may be administered under appropriate circumstances to produce the toxin in situ. Once identified, such toxins produced by soil-dwelling bacteria may have utility in diverse other areas as antifungal or antibiotic agents.
An example of a biological control agent into which significant scientific and economic development has occurred is Bacillus thuringiensis. B. thuringiensis strains produce toxic proteins which have the ability to specifically kill certain insects. Significant further research has identified a large number of B. thuringiensis strains having variations in target range and efficacy. In addition, research has been conducted on methods for stabilizing and applying such toxins, or strains harboring them, to a wide variety of field crop situations. It was also discovered that knowledge of B. thuringiensis strains was largely transferable to new strains since the toxins required for biological control and methods for preparing inocula for use in the field were generally similar among strains.
B. cereus (Bc) is closely related to B. thuringiensis (Bt), which has been used for insect biocontrol for about 30 years. Bc and Bt are generally considered to be members of a single species and the names are artifacts of history (Ash et al., 1991a,b; Carlson et al., 1994; Gordon, 1975; Logan and Berkeley, 1984; Lysenko, 1983; Priest, 1993; and Zahner et al., 1989). The major difference between Bc and Bt appears to be the presence of plasmids in the latter that encode an insecticidal crystal toxin responsible for insect biocontrol by Bt. Bc is a known food contaminant that can cause diarrhea in humans. Outbreaks of Bc food poisoning are associated with improper food storage and handling and require ingestion of large numbers of Bc spores (Gilbert, 1979). There have been no reported outbreaks of food poisoning associated with Bt use, presumably because it does not reach the human food supply in sufficient populations to cause a problem.
Previously it has been found that a specific strain of Bacillus cereus, which has been referred to both as UW85 and by its ATCC designation 53522, has biocontrol efficacy in many applications. See, e.g., U.S. Pat. No. 4,877,738. UW85 has proven to be an effective biocontrol agent of damping-off and root-rot diseases of soybeans and alfalfa under diverse field conditions in the upper Midwest U.S. (Handelsman et al., 1990; Osburn et al., 1995). The UW85 B. cereus strain protects alfalfa seedlings from damping off caused by Phytopthora medicaginis (Pmm), tobacco seedlings from Phytopthora nicotianae, cucumber fruits from rot caused by Pythium aphanidermatum, and peanuts from Sclerotinia minor. In a five-year field study on soybeans, UW85 applied either on the seed or in-furrow increased soybean yields significantly in all five years at a site with high disease pressure; yield enhancements were typically in the range of 35% to 139% depending on the severity of disease. In each year, the magnitude of the yield effect of UW85 was similar to that of Ridomil, the in-furrow formulation of metalaxyl, which controls Oomycete pathogens (Osburn et al., 1995). UW85 has been extensively field-tested and is currently in review by EPA for registration as a seed treatment for various crops.
UW85 produces two antifungal compounds that independently contribute to suppression of damping-off fungi. The more potent of these compounds, is zwittermicin A, a novel aminopolyol (Silo-Suh et al., 1994; He et al., 1994). Other, diverse strains of B. cereus found in all tested soils also suppress plant disease and produce zwittermicin A (Stabb et al., 1994; Raffel et al., 1996). Thus, this species has broad potential for biocontrol applications. The second compound is kanosamine, a well known aminoglycoside antibiotic.
Inoculation with B. cereus appears to have a negligible long-term impact on the environment. This species is common and present in all soils we have tested at approximately 104 cfu/gm soil (Stabb et al., 1994; Raffel et al., 1996). Data suggest that roots have a carrying capacity for Bc since by the end of the growing season, soybean roots derived from seeds treated with Bc UW85 had the same populations of Bc as untreated roots. However, the treated roots carried UW85 whereas untreated roots carried indigenous Bc strains (Halverson et al., 1993). Therefore, it appears that when UW85 is applied to seeds, it colonizes the roots of the plant at low populations and then dies off to the background level by the end of the season, suggesting that it will have minimal environmental impact. We have shown that early in the season, treatment of seeds with UW85 can result in a change in the microbial community on roots (Gilbert et al., 1993), but this effect does not seem to be sustained beyond the first 10 days (Raffel and Handelsman, unpublished). Overall, the existing data suggest that seed treatment with UW85 is a benign environmental event since Bc is a common soil bacterium and UW85 does not maintain high populations for long, which is in concert with other studies of Bacillus (Kim et al., 1997).
There is significant interest in developing numerous strains of Bc as inoculants for disease suppression on field crops, fruit and vegetable crops, greenhouse and ornamental production, and turf management, and in choosing strains adapted to particular host species or geographic locations. Other such strains have been identified. Thus, the invention disclosed herein will have application to many strains of Bc, not just to UW85 and the other strains noted herein.
Biocontrol has long been thought to be safer for the environment and human health than synthetic pesticides (Cook et al. 1996; Benbrook et al., 1996). As more bacterial biocontrol agents have reached the federal regulatory agencies for review, the relatedness of some of the bacteria and human pathogens has raised concerns by the agencies themselves and the public. For example, a recent registration application for a soil isolate of Burkholderia cepacia (Parke et al., 1991) raised concern from the cystic fibrosis community because members of this bacterial species are opportunistic pathogens of cystic fibrosis patients (Govan and Deretic, 1996).
There is some evidence to suggest that the B. cepacia biocontrol strains are genetically and physiologically different from the human isolates, but the risks associated with use of these bacteria remain in question. This type of issue is likely to emerge more frequently as biocontrol research yields a greater diversity of strains. Many of the strains being developed for biocontrol are common soil bacteria, and many soil bacteria are, or have close relatives that are, human pathogens. It is therefore essential to assess the environmental exposure associated with use of these organisms and determine whether that exposure is greater with biocontrol practices than with standard agricultural practices. Furthermore, there is an excellent opportunity to use the powerful techniques of genetic engineering to make biocontrol organisms safer by deleting genes associated with unwanted activities. This will have two impacts: providing biocontrol agents that pass regulatory review, and providing the public with examples of genetic engineering used to make the food supply safer.
Despite the good safety record of Bt, lingering concerns remain about registration of products containing Bt or Bc. Many strains of Bc and Bt produce an enterotoxin, denoted HBL, that is associated with mammalian toxicity (Beecher and Macmillan, 1991; Beecher et al., 1995). All of the major Bt strains on the commercial market produce HBL (Damgaard, 1995) and, as a result, the EPA has expressed hesitation about future registration of Bt strains. Bc is a recognized food toxicant and therefore must be demonstrated to be safe before it is used as an inoculant. There is strong evidence that HBL is responsible for the toxicity of most pathogenic Bc strains because purified HBL mimics Bc toxicity and there is a high correlation between toxicity and HBL production among Bc strains. The two key issues with Bc inoculant strains are the presence of the gene for enterotoxin production and any associated potential for human exposure to Bc from food grown from Bc-treated seed. It is not known whether other members of the so-called Bacillus cereus group, namely the B. anthracis strains, produce the enterotoxin, although both B. cereus and B. thuringiensis do produce the enterotoxin.
HBL is a tripartite enterotoxin, that includes a binding component, B, and two lytic components, L1 and L2 (Beecher and Macmillan, 1991). All three components are absolutely necessary for enterotoxigenic activity, as assessed by fluid accumulation in the ligated rabbit ileal loop assay and vascular permeability, which are currently the best animal models for Bc toxicity (Beecher et al., 1995; Glatz et al., 1974).
The genes encoding each of the components have been cloned and sequenced, and appear to be cotranscribed in an operon in the order L2, L1, B (Heinrichs et al., 1993; Ryan et al., 1997). Studies indicate that HBL production is a common trait among strains of B. cereus isolated from a variety of sources, including flour, rice, dairy products, and soil (Carlson et al., 1994; Damgaard et al., 1996; Granum et al., 1993; Lee et al., 1995; Mosso et al., 1989; Te Giffel et al., 1997; Yusuf et al., 1992).
A PCR screen of nearly 175 soil and root isolates for hblA using primers derived from the sequence of the hblA gene published by Heinrichs et al., 1993 showed that approximately 70% contain hblA, the gene that encodes the B component (Raffel and Handelsman, unpublished). UW85 contains this gene and produces the toxin when grown in tryptic soy broth as shown by ELISA with antiserum against HBL.
In addition to being the most important and widespread toxin in Bc, HBL is the only known mammalian toxin in strain UW85. In addition to being important biologically, HBL is important from a regulatory perspective. EPA requests data about the production of HBL (and not about other toxins) prior to registration of Bc and Bt strains.
What is lacking in-the art is a strain in the Bacillus cereus group having utility as a biocontrol agent that is also deficient in the HBL enterotoxin. The present invention responds to that regulatory concern as well as the scientific evidence that HBL is important in mammalian toxicity by Bc.
The present invention is summarized in that a mutant Bacillus strain is deficient in the enterotoxin HBL. The invention is further summarized in that the mutant strain is obtained by altering at least one of three polynucleotide sequences that otherwise would encode a component of the HBL enterotoxin, so that the polynucleotide sequence can no longer encode a functional component and, therefore, an active HBL enterotoxin cannot be produced.
The present invention is further summarized in that a genetic construct for use in accordance with the present invention contains an allele of a polynucleotide sequence that encodes at least one inactive component of HBL enterotoxin.
It is an advantage of the present invention that the enterotoxin can be eliminated from the Bacillus without adding any new DNA in the organism. Therefore, under the current EPA definition of genetic engineering (Federal Register 1997, 17910-17958), the resulting mutants are not considered genetically engineered and will not be subject to any regulations that do not apply to the wild type strain.
Other objects, advantages and features of the invention will become apparent upon consideration of the following detailed description taken in conjunction with the accompanying drawings.